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1Arizona Research Laboratories Division of Neural Systems, Memory and Aging, Departments of 2Psychology and 3Neurology, 4Evelyn F. McKnight Brain Institute, University of Arizona, Tucson, Arizona
Submitted 21 November 2007; accepted in final form 10 April 2008
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ABSTRACT |
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INTRODUCTION |
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; Foster 2007; Penner and Barnes 2007; Wilson et al. 2006). Synaptic transmission at the perforant path-granule cell synapse undergoes a number of changes during aging. These changes include a reduction of the number of synaptic contacts in the termination zone of the perforant path during aging (for review, see Geinisman et al.. 1995) and a larger excitatory postsynaptic potential (EPSP) in aged rats (after adjusting for presynaptic fiber potential amplitude) that decreases as a function of age (Barnes and McNaughton 1980). Furthermore, aged rats show a larger granule cell spike discharge for a given EPSP amplitude (Barnes 1979) together with a larger unitary EPSP (McNaughton et al. 1981) and larger quantal content (Foster et al. 1991) of perforant path synapses. In the dentate gyrus the non-NMDA-mediated field EPSP (fEPSP) increases during aging, whereas the NMDA receptor-mediated fEPSP decreases (Rao et al. 1994). Last, the threshold to induce long-term potentiation (LTP) in the dentate gyrus of aged animals is higher compared with young animals (Barnes et al. 2000), and LTP decays more rapidly over the course of days in this part of the hippocampus in aged rats (Barnes 1979). Some of these electrophysiological changes observed in old animals might be interpreted as compensatory responses to loss of synaptic contacts, although the faster rate of LTP decay at granule cell synapses is correlated with age-related memory deficits (Rosenzweig and Barnes 2003).
While field potential recordings in the molecular layer of dentate gyrus have yielded larger non-NMDA/NMDA fEPSP ratio in aged rats compared with young (Rao et al. 1994), a confirmation of these findings at the level of evoked ionotropic glutamatergic currents is still missing. Thus the present study was designed to determine whether and how individual components of the excitatory ion currents at the perforant path-granule cell synapse change during aging. To do this, we recorded excitatory postsynaptic currents (EPSCs) from granule cells using a standard Cs+-based internal solution in the patch pipettes. The responses were evoked by stimulation of the perforant path that arises from layer II entorhinal cortical cells (Steward and Scoville 1976) in slices obtained from young and aged Fischer-344 rats.
In recent years evidence has accumulated for the expression and function of voltage-sensitive ion channels expressed in dendrites of central neurons (Johnston et al. 1996; Magee 2000; Stuart et al. 1997). To test whether active dendritic conductances such as the cationic current Ih (Bender et al. 2007; Chen 2004; Poolos et al. 2002) or inwardly rectifying potassium currents (Karschin et al. 1996; Koyrakh et al. 2005; Ponce et al. 1996), which are blocked in whole cell recordings by intracellular Cs+, alter synaptic potentials in an age-dependent manner (Bender et al. 2001; Magee 1999), we also recorded fEPSPs and population spikes in the granule cell layer of young and old rats in the absence and presence of extracellular Cs+. For both sets of experiments, spatial learning and memory deficits were confirmed in aged animals by testing the young and old rats in the Morris water maze prior to the electrophysiological recordings. Portions of these results have been published previously in abstract form (Houston et al. 2004; Krause et al. 2002).
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METHODS |
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Animals
For experiment 1, we used 11 young (mean age: 3.0 mo at the time of behavioral testing) and 11 aged (mean age: 22.2 mo at the time of behavioral testing) male Fischer-344 rats. For experiment 2, we used six young (mean age: 4.0 mo at the time of behavioral testing) and six aged (mean age: 26.0 mo at the time of behavioral testing) male Fischer-344 rats. All animals were obtained from the National Institute of Aging colony at Harlan (Indianapolis, IN). The time between the behavioral and electrophysiological testing was 
2 wk. None of the included rats had cataracts or other visual or motor problems that prevented them from good performance on cued versions of the swim task. Rats were housed in pairs and had access to food and water ad libitum. All experiments were conducted during the dark phase of their 12-h light/12-h dark cycle. After behavioral testing in experiment 1, animals were shipped to the Max-Planck-Institute for Experimental Medicine in Göttingen, Germany, for the whole cell patch-clamp experiment for electrophysiological testing described in the following text. For experiment 2, rats were shipped, following behavioral testing, to Tensor Biosciences in Irvine, CA, for the field potential recording experiment examining the effects of extracellular Cs+ described in the following text. In each experiment, rats were allowed to accommodate to their new environment for 
1 wk after arrival.
Morris water maze
To assess the rats' spatial learning ability, we tested all animals in a 185-cm-diam pool essentially as previously described (Shen and Barnes 1996). Procedures used for water maze training in experiments 1 and 2 were identical except that in experiment 2, the rats' movements were recorded and analyzed with a camera mounted over the pool and computer software (HVS Image). These data were used to obtain the corrected integrated path length (CIPL) in addition to the latency to find the platform. Because aged animals swim more slowly than do young animals, CIPL is a preferred measure to test the animals' spatial memory because it is independent of the animals' swim speed; however, for experiment 1, the tracking data were lost due to a technical error and thus only latency measures are available.
During the spatial (hidden-platform) version of the swim task, a circular escape platform was present in a constant location, submerged 1 cm below the water surface. Each rat performed three training blocks per day (2 training trials per block) for 4 days (24 trials total) with the release location varied randomly from trial to trial. Rats were allowed a 60-s intertrial interval within a given training block and 30–60 min of recovery time between training blocks, where rats were in a warm chamber so that body temperature was maintained.
Animals were also given a probe trial, which immediately followed the last spatial trial on day 4 of the spatial task. For the probe trial, the platform was removed from the tank, and the rats were allowed to swim and search for the platform for a total of 60 s (data for this procedure are only available for experiment 2).
To control for possible age-related deficits in visual acuity and swimming ability, the same rats were also trained on a second version of this task in which a visible platform was moved randomly to one of four locations in the tank after each trial (cued version). A total of 12 visible-platform trials were performed (6 trials daily for 2 days). In all trials, we recorded the time to reach the platform.
In vitro hippocampal slice preparations
PATCH-CLAMP RECORDINGS.
Animals were deeply anesthetized with halothane (Hoechst) and subsequently decapitated. The brain was quickly removed and submerged in ice-cold oxygenated artificial cerebrospinal fluid (ACSF) containing (in mM) 125 NaCl, 1.25 KCl, 1.25 KH2PO4, 1.5 MgCl2, 25 NaHCO3, 1 CaCl2, and 10 D-glucose (pH 7.4). Transverse hippocampal slices were prepared with a vibratome (Leica; 400 µm thick). The slices were held in CSF and a mixture of 95% O2-5% CO2 for 
6 h at room temperature. After 1 h incubation, individual slices were transferred to a submerged chamber and kept in place by a light grid made out of nylon stocking for electrophysiological recording.
FIELD POTENTIAL RECORDINGS.
Animals were deeply anesthetized with halothane (Sigma) and subsequently decapitated. Transverse slices (400 µm thick) were obtained from the dorsal hippocampus with a tissue chopper. Slices were incubated and recorded from at 32°C in an interface-style chamber in ACSF solution containing (in mM) 124 NaCl, 3 KCl, 1.25 NaH2PO4, 3.75 MgSO4, 25 NaHCO3, 2 CaCl2, and 10 glucose, gassed with 95% O2-5% CO2 (pH 7.4). A warm, humidified mixture of 95% O2-5% CO2 was added to the chamber throughout recovery and recording periods. Slices were allowed to recover for a period of 1 h before recordings began.
Electrophysiological recordings, stimulation, and data acquisition
PATCH-CLAMP RECORDINGS.
During recordings from hippocampal slices the [Ca2+] was raised to 2.5 mM, and 20 µM (–)-bicuculline-methochloride (Research Biochemicals International) was added to block GABAA receptor-mediated currents (flow rate: 2 ml/min). In some experiments, we added 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or D-AP5 (Tocris) to the bath solution to block AMPA/kainate- and NMDA-mediated currents, respectively. Patch pipettes (3–5 M
) were pulled (Narishige PP-830) from borosilicate glass (0.9 mm ID, 1.5 mm OD, Hilgenberg) and were filled with a solution containing (in mM) 115 CsCl, 20 tetraetylammonium chloride (TEA), 5 NaCl, 4 Mg-ATP, 10 HEPES, 5 EGTA, 5 QX-314, and 0.4 GTP (titrated to pH 7.3 with CsOH; osmolarity: 300 mOsm). In this solution, potassium currents and Ih were blocked by Cs+ and TEA, and voltage-sensitive sodium channels were blocked by QX-314 (Calbiochem). The liquid junction potential was –4 mV and was not subtracted from the command voltages. Dentate granule cells were identified by their location in the slice, patched using the "blind method" (Blanton et al. 1989), and recorded in the whole cell configuration. Granule cells were further identified by the presence of synaptically activated glutamatergic currents on afferent stimulation of the perforant path. Synaptic currents (60% of maximal amplitude) were evoked once every 10 s by orthodromic stimulation of the perforant path (100 µs duration) with 70–300 µA stimulation intensity (Isoflex, AMPI), using bipolar stainless steel electrodes (distance between tips 200 µm). The distance between the stimulation electrode and the recorded cell was 200–250 µm. The majority of the recordings were made in the dorsal (or attached) blade of the dentate gyrus. Synaptic currents were low-pass filtered at 1 kHz, amplified with a patch-clamp amplifier (EPC-9, Heka), and digitized at 4 kHz using the software package Pulse (Heka). In all recordings, series resistance was regularly monitored by –5-mV test pulses of 100-ms duration at –70-mV holding potential (sampled at 40 kHz). Series resistance was estimated from the current difference between the transient peak at the beginning and the steady state at the end of that pulse. Average series resistance was 12.4 ± 0.7 M in the young animals and 16.4 ± 1.0 M in the aged animals, respectively. Only recordings with series resistances <25 M and changes no more than 15% were accepted. The input resistance was estimated from the 100-ms pulse by measuring the current difference prior to the pulse and at steady state near the end of the test pulses. Between one and five neurons were recorded from each animal, but data were averaged for a given animal to use for the age comparisons, and to prevent introducing a sampling bias. All recordings were made at room temperature (20–22°C).
FIELD POTENTIAL RECORDINGS.
One or two slices were recorded from each animal. Input-output relationships were obtained by stimulating the perforant path (80-µs pulse width, 10–50 µA stimulation intensity) with a bipolar stimulation electrode. Excitatory postsynaptic field potentials (fEPSPs) were recorded with a glass pipette filled with 2 M NaCl (2–4 M), which was placed near the granule cell layer/hilar border. Maximal population spike (PS) amplitudes were initially ascertained. Five other intensities were determined, the lowest being set at 1/5 the intensity of the maximal response and the other four set at equal intervals between this maximum and minimum. Three sample responses were recorded at each intensity (sampled at 20 kHz) with an overall stimulation rate of once per 30 s. Each slice was first tested in ACSF, followed by the same protocol in 2 mM CsCl, which was added to the perfusion medium (1 ml/min flow rate). If not otherwise specified, all chemicals used were purchased from Sigma.
Data analysis
WATER MAZE. To analyze latency and corrected integrated path length in the hidden platform and visible platform task, a two-way repeated-measures ANOVA was used with age (young vs. aged) as the between-subjects factor and training (24 trials) as the repeated within-subjects factor. Post hoc tests were used if appropriate. Rat's dwell times in the four quadrants of the pool during the probe task were analyzed using one-way ANOVA. Fisher's protected least-significant difference post hoc tests were used to determine whether rats spent significantly more time in the target quadrant over other quadrants.
WHOLE CELL PATCH-CLAMP RECORDINGS.
To establish current-voltage relationships (I-V relationship), the maximal amplitudes were measured (between 6 and 14 ms after stimulation onset, representing non-NMDA-mediated currents) and the amplitudes 70–80 ms after stimulation onset (representing NMDA-mediated currents) were obtained at every holding potential of the averaged waveforms (Fig. 3C). Current waveforms shown are averages of 5–10 single samples. The time constants of current onsets or decays were calculated using a single exponential fit. To estimate the positive slope conductance of non-NMDA- and NMDA-mediated currents, a line (least-square method) was fit to the averaged current amplitudes at holding potentials –10, 10, 30, and 50 mV. Regression coefficients (Pearson) were 0.93, and correlations of all line fits were significant (P < 0.05). Student's t-test were used for comparisons of two means, and alpha was set at the 0.05 level. For data analysis, the programs PulseFit (Heka) and Igor (Wavemetrics) were used.
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RESULTS |
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BEHAVIORAL TESTING IN THE MORRIS WATER MAZE. In the spatial learning portion of the water maze task, a significant main effect of age was found [F(1,20) = 23.27, P < 0.0001], indicating that young animals reached the platform faster than did aged animals. A significant main effect of training was also found [F(23,460) = 15.47, P < 0.0001], indicating that the time to reach the platform was reduced with training. There was also a significant interaction of age and training [F(23,460) = 2.19, P < 0.001], suggesting that young animals benefited significantly more from the training than did the aged animals (Fig. 1A). A comparison of latencies of young and aged rats on each day of training shows that the young animals were significantly faster on any given day [day 1: t(112) = 3.35, P < 0.0001; day 2: t(112) = 5.19, P < 0.0001; day 3: t(112) = 9.62, P < 0.0001; day 4: t(112) = 6.77, P < 0.0001].
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Dentate gyrus granule cell EPSCs and non-NMDA/NMDA ratios in young and aged rats
To study the influence of aging on synaptic transmission in hippocampal granule cells, excitatory postsynaptic currents (EPSCs) were recorded in the whole cell configuration. Currents were evoked by orthodromic stimulation of the perforant path from young (27 neurons) and aged (24 neurons) rats. No differences were detected in several basic electrophysiological measures such as input resistances, current rise and decay time constants, reversal potentials, and absolute current amplitudes (Table 1, P > 0.05 in all cases). EPSCs evoked at different holding potentials were overall similar in both age groups and comparable to those recorded in very young (20–30 days) animals (see examples in Fig. 2) (Keller et al. 1991).
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The relationship between non-NMDA and NMDA-mediated currents in young and aged animals were determined by constructing current-voltage plots (I-Vs, Fig. 4). The currents were separated on the basis of their kinetics as described earlier. I-V relationships were linear (correlation coefficient r > 0.93; P < 0.05) for both age groups from holding potentials positive of –10 mV showed a positive slope conductance (Fig. 4A). The age effects observed were: the absolute values of slope conductances for non-NMDA currents were 3.29 ± 0.58 nS for young and 1.55 ± 0.20 nS for aged animals [t(20) = 2.75, P < 0.05], and the absolute values of slope conductances for NMDA currents were 2.10 ± 0.35 nS for young and 1.14 ± 0.14 nS for aged animals [t(20) = 2.53, P < 0.05; Fig. 4B]; the ratio of non-NMDA/NMDA slope conductance was 1.54 ± 0.05 for young and 1.34 ± 0.04 for aged animals, and was statistically different [t(20) = 2.90, P < 0.01; Fig. 4C].
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Experiment 2
The results from whole cell patch-clamp recordings in dentate gyrus granule cells in experiment 1 indicate a significant decrease in the non-NMDA/NMDA current ratio in aged animals. On the other hand, field potential recording experiments have suggested that there is an increase in this ratio with increasing age (cf. Yang et al. 2008). One potential difference between the two recording methods is the fact that the patch-clamp recordings were conducted with Cs+-containing patch pipettes to aid in isolating glutamatergic currents, whereas no Cs+ was present in the conditions used for field potential recording. To test the hypothesis that the Cs+ could play a role in the differences observed between studies, field potentials were recorded in young and old memory-characterized rats in the presence and absence of extracellular 2 mM Cs+. Intracellular Cs+ blocks Ih and several potassium currents (Coetzee et al. 1999). When applied extracellulary, Cs+ blocks Ih (Chen 2004; Maccaferri et al. 1993) and inwardly rectifying currents (Lesage et al. 1995), such as G-protein coupled inwardly rectifying potassium channels (GIRKs; Halliwell and Adams 1982). The HCN channels (Ludwig et al. 1998; Santoro et al. 1998) underlying the Ih, are expressed in hippocampal granule cell dendrites (Bender et al. 2007), and can shape dendritic EPSPs (Magee 1998–2000; Poolos et al. 2002; Williams and Stuart 2000). The field potential recordings allowed assessment of the effects of Cs+ on both the synaptic and population spike components of the evoked responses. The rationale for experiment 2 was thus to determine whether the effect of the presence or absence of Ih or inwardly rectifying currents would produce effects on the fEPSP and PS that could explain the difference between the results obtained in the present experiment using patch-clamp methods versus those obtained using extracellular field potential recording methods.
Behavioral testing in the water maze
HIDDEN PLATFORM TEST. There was a significant main effect of age in the hidden platform, spatial version of the swim task. Young rats learned to take a more direct route to the hidden platform over trials than did the old rats [F(1,10) = 35.95; P < 0.0001]. In addition, there was a significant effect of training [F(23,230) = 5.01; P < 0.0001] and an interaction effect [F(23,230) = 1.84; P < 0.013], indicating that the two groups did not benefit equivalently from the training sessions (Fig. 5A).
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VISIBLE PLATFORM TEST. To test the possibility that the age difference in performance on the hidden platform test was a result of visual impairments, a visible platform test was conducted. There was no significant main effect of age between groups in this test [F(1,10) = 7.55; P = 0.21]. There was, however, a significant effect of training [F(11,110) = 2.59; P < 0.006] with no significant interaction [F(11,110) = 1.07; P = 0.39], indicating that there was no difference detected between groups in the improvement of their navigational accuracy toward the platform over trials (Fig. 5C).
Differential effects of extracellular Cs+ on dentate fEPSPs in young and aged rats
At least 1 wk following behavioral testing, the slice electrophysiology experiments were initiated. fEPSPs and PSs in the dentate granule cell layer were measured before and after extracellular bath application of Cs+ (2 mM). Seven slices from young and eight slices from aged animals were investigated (6 animals in each group). Figure 6A shows typical examples of responses obtained from young and aged animals during recordings of field potential responses at several different intensities. To ensure that the stimulation and recording procedure by itself did not alter the response characteristics, the experiments were begun by obtaining responses at a low intensity up to the highest intensity and then again back down to the lowest intensity. For each age group, the fEPSP slope and PS area obtained under the increasing or decreasing intensity protocols were not different. Thus this stimulation paradigm per se did not change response magnitudes.
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0.005] and intensity [F(4,59) = 2.72, P 0.05] for fEPSP slopes. Using two-way ANOVA for the PS area, we found significant main effect of intensity [F(4,59) = 5.22, P 0.005] but not for age [F(1,59) = 2.10, P 0.15].
These data suggest that extracellular Cs+, which blocks mainly Ih in dentate gyrus granule cells (Chen 2004), affects field potentials differently in young and aged rats, possibly indicating different expression levels of the channel protein underlying this current, and/or a different age-dependent distribution of Cs+-sensitive channels in soma, proximal and distal dendrites.
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DISCUSSION |
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Glutamate receptor-mediated synaptic transmission in dentate granule cells of young and old rats
The data obtained in this study show that glutamatergic currents in dentate granule cells from adult and aged rats are very similar to those in 3- to 4-wk-old rats. Reported rise time constants of glutamatergic EPSCs for these very young rats are 0.5–1.9 ms for non-NMDA EPSCs and 4–9 ms for NMDA EPSCs (Keller et al. 1991) for hippocampal granule cells, and 0.5–4 ms for non-NMDA EPSCs and 4–8 ms for NMDA EPSCs in CA1 pyramidal cells (Hestrin et al. 1990; Otmakhova et al. 2002). These values do not substantially deviate from the values obtained from the mature and old hippocampal granule cells reported here. The reduced slope conductance in the I-V relationship of aged animals reported here is in agreement with smaller fEPSP amplitudes in aged animals (cf. Yang et al. 2008) and is expected given the loss of perforant path synaptic contacts during aging (Geinisman et al. 1995). This synaptic loss is accompanied by a reduction in ionotropic glutamate receptor mRNA and protein expression in the hippocampus of aging rodents (Barnes and McNaughton 1980, 1983; Clark et al. 1992; Geinisman et al. 1995; Lippa et al. 1981; Magnusson 2000; Magnusson and Cotman 1993; Nicolle et al. 1996; Pagliusi et al. 1994; Tamaru et al. 1991; Wenk and Barnes 2000).
Patch-clamp recording experiments are typically conducted on animals at substantially younger ages than those in the present experiments. Because of the greater challenge of applying these methods for use in mature tissue, it was important to establish appropriate voltage control in these hippocampal granule cells. Thus the series resistance in the present study was carefully monitored to assess electrical access to the inside of the cell. In addition, intracellular Cs+ and the sodium blocker QX-314 was used to increase the input resistance by blocking several ion channels. The median input resistance was 520 M for young and 600 M for aged animals. These values are, in fact, similar to values obtained by other investigators when using Cs+-containing patch pipettes in young animals (Stabel et al. 1992). Given that the highest accepted series resistance was 25 M in the present study, the maximal voltage error near the soma was 7% but was <5% for the majority of the recordings. A larger voltage error in the distal dendrites, however, cannot be excluded due to limited space-clamp conditions. Nonetheless, current-clamp recordings using sharp electrodes, where access resistance plays no role, does reveal very similar results to those obtained in the present study. In agreement with the present study, no differences in resting membrane potential, input resistance or action potential threshold in granule cells of young and old Fischer-344 rats were detected (Barnes and McNaughton 1980).
Reduction of glutamatergic currents at the perforant path–granule cell synapse during aging
Pharmacological characterization indicates that non-NMDA (AMPA and kainate) and NMDA receptor-mediated currents are the two main components of the EPSCs evoked by perforant path stimulation. Ratios of these currents have been studied in several brain regions such as neocortex, hippocampus, and ventral tegmental area and are generally considered to be a dynamic measure of synaptic modification (Heynen et al. 1996, 2000; Otmakhova et al. 2002; Sjostrom et al. 2007; Sprengel et al. 1998; Ungless et al. 2001). Several methods are available to establish measures of a non-NMDA/NMDA response ratio in single neurons: 1) a pharmacological method, where a neuron is held at +40 mV and first recorded in normal ACSF and subsequently in the presence of APV (an NMDA receptor blocker). This method allows separation of the dual-component EPSC (Ungless et al. 2001), 2) an alternative pharmacological method, where the charge at –20 mV holding potential in normal ACSF after application of APV is measured (Otmakhova et al. 2002); and 3) a biophysical method, where an I-V relationship is recorded and the slope conductance for each receptor subtype is measured on the basis of the kinetics of the individual component (Sprengel et al. 1998). The latter biophysical method was preferred in the present study as a potential control for the possibility of changing series resistances before and after administration of glutamatergic blockers. Although several groups have reported results of non-NMDA/NMDA ratios, different recording conditions and analyses along with different species do not allow direct and meaningful comparisons of these ratios and thus will not be attempted here. We can confidently conclude, however, that under the whole cell recording conditions of the present study, non-NMDA and NMDA EPSCs decline with age in hippocampal granule cells, and this decline occurs in aged rats with hippocampal-dependent learning and memory deficits.
Role of Cs+-sensitive currents in synaptic transmission in dentate gyrus
Results from the present study illustrate a decline in glutamatergic EPSCs (Fig. 4B) and smaller dentate granule cell fEPSP slopes and PS areas (Fig. 6, B and C) in aged, learning- and memory-impaired rats consistent with previous studies (Barnes 1979; Barnes and McNaughton 1980; Yang et al. 2008). The increase in the non-NMDA-mediated portion of the fEPSP in aged animals in the Yang et al. study is not congruent with the smaller non-NMDA/NMDA ratio observed in the whole cell recording data collected in experiment 1 of the present study. There are a number of technical differences between these preparations that may explain this apparent discrepancy. Among these is the fact that the patch pipette contained blockers for sodium currents, Ih, and several potassium currents. None of these currents were blocked in the fEPSP recordings reported in the Yang et al. (2008) study. Internal Cs+ blocks Ih and inwardly rectifying potassium currents (e.g., GIRKs) in granule cells (Chen 2004; Mellor et al. 2002). Both HCN channels, which underlie Ih, and GIRKs are expressed in the dentate gyrus (Karschin et al. 1996; Monteggia et al. 2000; Moosmang et al. 1999; Ponce et al. 1996; Santoro et al. 2000). Functionally, these currents stabilize the neuron's resting membrane potential and could play a role in the size of fEPSPs and overall excitability. Particularly in dendrites, Ih (Berger et al. 2001; Magee 1998, 1999; Poolos et al. 2002) and GIRKs (Takigawa and Alzheimer 1999, 2002, 2003) attenuate excitatory input. The activation time of these currents are on a similar time scale to that of dendritic fEPSPs (Magee 1998). Thus amplitude and/or time course of the fEPSPs recorded in the molecular layer of the dentate gyrus may be differentially affected by an age-dependent expression of Ih and GIRKs. The data obtained in the present report are at least consistent with this hypothesis. Our results indicate a stronger attenuation of fEPSPs in young rats by Cs+-sensitive currents. This could be due to higher expression of these channels and/or a shift of their activation curve to more depolarized potentials in young animals (e.g., in the case of Ih, the results might be explained by higher cAMP levels in younger animals) (Beaumont and Zucker 2000; Pedarzani and Storm 1995; Tombaugh et al. 2005).
Although our data implicate changes in Cs+-sensitive potentials as an explanation for the differences observed between the present study and the Yang et al. (2008) results, we cannot entirely exclude other possible contributing factors. Specifically, the age-related changes observed in the non-NMDA/NMDA receptor ratios appear to be dependent on whether field potential recording methods were used to establish the ratio (see Yang et al., 2008) or whether whole cell patch-clamp recordings were used as in the present study. Besides the presence of cesium in the patch pipette, other possible technical explanations for the differences in results between these studies include the fact that the whole cell recordings were performed in a submerged chamber while the field potential recordings used an interface chamber, the recording temperature was different (22°C in field potential recordings), there were minor differences in ACSF composition (CA2+ was slightly higher in whole cell recording, and [K+] and [Mg2+] was slightly higher in the field potential recordings), and during whole cell recordings there was the possibility of wash out of intracellular constituents. While these differences may have contributed to the differences between the data observed here and the Yang et al. (2008) data, it is likely that Cs+-sensitive currents remain a major explanatory factor.
In summary, the present data suggest that aging not only changes expression and function of ionotropic glutamate receptor-mediated currents but also results in changes of the dendritic integration of excitatory inputs.
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GRANTS |
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ACKNOWLEDGMENTS |
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Present addresses: M. Krause, Ernest Gallo Clinic and Research Center, Emeryville, CA 94608; Z. Yang, EnVivo Pharmaceuticals, Watertown, MA 02472; G. Rao, Department of Neurobiology and Anatomy, W. M. Keck Center for the Neurobiology of Learning and Memory, University of Texas Medical School at Houston, Houston, TX 77225.
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FOOTNOTES |
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Address for reprint requests and other correspondence: C. A. Barnes, University of Arizona, Evelyn F. McKnight Brain Institute, Life Sciences North Bldg., Rm. 384, Tucson, AZ 85724 (E-mail: carol{at}nsma.arizona.edu)
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